Risk factors, early detection and treatment of neuropathy in leprosy

Transcription

1 Risk factors, early detection and treatment of neuropathy in leprosy Risicofactoren, vroege opsporing en behandeling van neuropathie in lepra Inge Margriet Wagenaar

2 2016 Inge Margriet Wagenaar ISBN: Printing: GVO, Ede No part of this thesis may be reproduced, stored in a retrieval system or transmitted in any form or by any means, without the prior permission of the author or, when appropriate, of the publishers of the publications. The studies presented in this thesis were funded by The American Leprosy Mission, the German Leprosy and TB Relief Association, the Netherlands Leprosy Relief, the Ordre de Malte, and the Turing Foundation. This thesis was printed with financial support of the Department of Public Health, Erasmus Medical Centre, and of the Erasmus University Rotterdam.

3 Risk Factors, Early Detection and Treatment of Neuropathy in Leprosy Risicofactoren, vroege opsporing en behandeling van neuropathie in lepra Proefschrift ter verkrijging van de graad van doctor aan de Erasmus Universiteit Rotterdam op gezag van de rector magnificus Prof.dr. H.A.P. Pols en volgens besluit van het College voor Promoties. De openbare verdediging zal plaatsvinden op woensdag 2 november 2016 om uur door Inge Margriet Wagenaar geboren te Wageningen

4 Promotiecommissie Promotor: Prof.dr. J.H. Richardus Overige leden: Prof.dr. E.P. Prens Prof.dr. P.A. van Doorn Prof.dr. A. Geluk Copromotor: Dr. J.W. Brandsma

5 Voor oma Margriet

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7 Contents I Introduction Chapter 1 General introduction 9 II Methodological studies on early detection of Nerve Function Impairment Chapter 2 Chapter 3 Two randomized controlled clinical trials to study the effectiveness of prednisolone treatment in preventing and restoring clinical nerve function loss in leprosy: the TENLEP study protocols 29 Normal threshold values for a monofilament sensory test in sural and radial cutaneous nerves in Indian and Nepali volunteers 49 Chapter 4 Reliability of Clinical Nerve Function Assessment in Peripheral Neuropathies 67 Chapter 5 Early detection of neuropathy in leprosy: A comparison of five tests for field settings 77 III Treatment of and risk factors for leprosy Chapter 6 Effectiveness of 32 versus 20 weeks of prednisolone in leprosy patients with recent nerve function impairment: a randomized controlled trial 95 Chapter 7 Prednisolone Adverse Events in the Treatment of Leprosy Neuropathy 119 Chapter 8 Diet-related risk factors for leprosy: A case-control study 133 IV Discussion and summary Chapter 9 General discussion 155 Summary 171 Samenvatting 175 Dankwoord 179 Curriculum Vitae 181 List of publications 183 PhD portfolio 185

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9 General introduction Chapter 1

10 1. General introduction into leprosy i. Leprosy Leprosy is an infectious disease that affects the skin, nerves and eyes and is caused by the Mycobacterium leprae. Even though the disease has been well curable since the introduction of multidrug treatment (MDT) in the early 1980 s, leprosy remains a public health problem in different parts of the world. Skeletal remains from 2000 B.C. discovered in India provide evidence that leprosy already exists for millennia. From India, the disease spread to other parts of the world [1], including to Europe where it reached its peak during the 13 th and 14 th century [2]. Leprosy disappeared from Europe in the eighteenth century, even before there was any treatment for leprosy available. Leprosy is now only known as a disease of the poor, and is mostly prevalent in low resource countries. The causative agent of leprosy, M. leprae, was discovered in 1873 by Armauer Hansen. It was the first time a bacillus was identified as a cause of disease in humans [3]. Even though leprosy is one of the oldest diseases known to man, there is still an incomplete understanding on the mode(s) of transmission, the mechanisms behind the immune response, nerve damage and reactions and unfortunately so far no effective vaccine has been developed to prevent clinical leprosy. Studying M. leprae is complex, since the organism has never been successfully cultured in vitro. ii. Symptoms and diagnosis Hypopigmented skin lesions are often the first symptoms of leprosy [4], however, due to low awareness or fear of stigma patients often wait to seek medical help until they experience more advanced symptoms caused by nerve function impairment, such as numbness or muscle weakness, [5,6]. The diagnosis for leprosy can be made when at least one of the three following cardinal signs is present: enlarged peripheral nerves, diminished touch perception in skin lesions or bacilli in skin smears. From skin smears or biopsies the bacterial index (BI) is derived which expresses the density of M. leprae on a logarithmic scale from 1 to 6 [7]. This can be used for diagnosis and to determine the effectiveness of therapy. iii. Transmission The incubation time of leprosy is usually long with reported maxima of 20 years, but is in most cases between 2 and 5 years [8]. The long incubation time makes it difficult to determine when and under what circumstances the disease was contracted, and therefore transmission of leprosy is not well-understood. The main transmission route is presumed to be from person-toperson through nasal droplets via the upper respiratory tract [9], although abraded skin is considered a possible port-of-entry as well [8]. Furthermore, M. leprae has been found to survive in soil for 46 days [10], which could also be a factor in transmission. 10

11 Only patients with the lepromatous type are considered infectious (see 3.i), and they remain infectious until the start of treatment with multi-drug therapy (MDT). Being a contact of a leprosy patient increases the risk of contracting the disease, depending on the intensity and duration of the contact [8,11]. Household members of lepromatous patients have the highest relative risk: 8-10 times the risk of the general population. In non-endemic areas, transmission within households plays an important role [12]. iv. Treatment In the 1940 s, dapsone was discovered as treatment against M. leprae. However, the bacteria developed resistance against the monotherapy and therefore a multi-drug therapy was introduced in The MDT is provided for free by the World Health Organization (WHO) since The WHO advises a therapy of 6 months for paucibacillary (PB) leprosy, with a daily dose of dapsone and a monthly dose of rifampicin. Multibacillary (MB) leprosy patients should follow a treatment of 12 months with daily doses of clofazimine and dapsone, together with rifampicin plus a high clofazimine dose once a month [13]. In line with older WHO guidelines, in some countries 24 months of MDT is given to MB patients. v. Prevention Even though there is no effective vaccine against leprosy, the BCG vaccine against tuberculosis also protects against leprosy [14,15]. The efficacy differs between populations, as case-controls studies have shown that vaccine protection lays between 20 and 90% [14]. 11

12 2. Epidemiology i. Prevalence Between the introduction of MDT in 1982 and the early 2000 s, the leprosy prevalence dropped by 90% [16]. However, in the last decade the decrease in prevalence has stabilized. The prevalence is defined as the number of leprosy cases on treatment, and thus depends on which treatment guidelines are followed [17]. A more stable indicator is the new case detection rate (NCDR), which shows the number of new detected cases in one year per population. Elimination of leprosy, defined as less than 1 leprosy case per population, was reached in 2000 at global level, but this target has not been reached in many regions of leprosy endemic countries. In 2014, the total number of reported new leprosy cases was [18]. Eighty-nine percent of these new cases were concentrated in eight countries: Bangladesh, Brazil, Democratic Republic of Congo, Ethiopia, India, Indonesia, Nepal and Nigeria. India alone accounted for 59% of new detected leprosy cases in In total, 13 countries had a new case detection of >1000. Details of the eight countries with the highest number of new cases are shown in Table 1. The PB/MB ratio differs between countries. Leprosy is more frequently seen in males, the malefemale ratio is about 1.5 to 2:1 [19]. Also, leprosy is seen most often in the age group years [20], but the disease has been diagnosed in children as young as three weeks old [21]. Table 1 also contains the proportion of new patients with Grade 2 Disabilities i.e. visible impairments and/or deformities. This is a useful indicator of delayed detection of leprosy. Table 1- New case detection, prevalence and patient details for the eight countries with the highest new case detection rates in 2014 [18] Country New case detection Prevalence % MB % female % children % grade 2 disabilities India Brazil Indonesia Ethiopia NR Bangladesh Congo Nepal Nigeria

13 ii. Countries The TENLEP study described in this thesis took place in four of the countries listed in Table 1: India, Bangladesh, Nepal and Indonesia. The new case detection rate (NCDR) of these countries is shown in Figure 1, together with the location of the centres where the studies were carried out. In India (Figure 1.A), we worked together with two centres. The first is the Foundation for Medical Research (FMR), a research institute located in Mumbai, which was cooperating with a referral centre in Mumbai to include patients in our studies. The second is JALMA institute, a specialised governmental leprosy hospital located in Agra. Several studies within this thesis were done in Bangladesh (Figure 1.B), all in Nilphamari Leprosy Mission Hospital, a specialized hospital with multiple outpatient clinics in the Rangpur district. In Nepal (Figure 1.C), also two centres were involved. The first is Anandaban Leprosy Hospital, located near the capital Kathmandu, also run by The Leprosy Mission. They are holding weekly clinic days in Kathmandu. The second is Lalgadh Leprosy Services Centre, a more rural hospital, which is one of the busiest leprosy centres in the world. In Figure 1.D the centre in Indonesia is shown. We worked together with the dermatology department of Dr. Soetomo hospital in Surabaya. Most patients came from the island of Madura, and were visited monthly by the staff. 13

14 DBLM Nilphamari New Delhi JALMA Dhaka FMR A B C Jakarta Surabaya New case detection rate per >5 1-2 No data D Kathmandu Anandaban Lalgadh Figure 1- Country maps indicating the new case detection rate in 2014 per district per inhabitants and the location of the hospitals for (A) India, (B) Bangladesh, (C) Indonesia, and (D) Nepal 14

15 3. Immunology and leprosy reactions i. Immune response and classification Leprosy is classified in two ways, the Ridley-Joplin and the WHO classification. The five-group Ridley-Jopling system is based on the characteristics of skin lesions and bacterial load and is used for research and clinical purposes [22]. The patient s immune response to the bacilli determines which type develops. At one end of the spectrum, patients with a mild form of the disease, the tuberculoid (TT) form, can be found. These patients show a strong cell-mediated immune response and have few well-defined skin lesions and no or low bacteria counts. At the other, more severe end, the lepromatous (LL) patient s immune system has a weak or absent response to M. leprae, resulting in multiple macules and/or nodules with many bacilli present [23]. In between these two extremes, the majority of the patients are classified in one of the three unstable borderline forms: borderline tuberculoid (BT), borderline borderline (BB) and borderline lepromatous (BL). The cellular immunity becomes weaker the more one gets to the lepromatous side. The intensity of this response can change and therefore patients can switch between the three borderline types, although most patients will move towards the lepromatous side (downgrading). It is not always possible to carry out and examine a skin smear test under field circumstances, therefore the WHO advises to use a simpler two-group classification system in these cases. When a person has up to five skin lesions the disease is classified as paucibacillary (PB) and patients with more than five lesions fall under the multibacillary (MB) group [13]. The PB group contains TT and BT types and the MB group includes BT, BB, BL and LL types [24,25]. If a skin smear is positive the patient should always be treated as an MB case. Next to determining which leprosy type develops, immunology is also very important regarding the susceptibility for leprosy. Analyses of M. leprae DNA in nasal swabs show that up to 31% of the general population in endemic areas carry the bacilli in their nose [9,26-28]. However, it is assumed that less than 1% of people exposed to M. leprae will develop clinical leprosy [20,29]. This means that a vast majority of the population has a strong immune response, leading to containment and spontaneous clearing of the disease. The factors leading to the differences in immune response between persons are not completely understood, but genetics, nutritional status and socioeconomic status are thought to influence the host reaction to the leprosy bacilli [30-33]. ii. Reactions In part of the leprosy patients, the antigens of the (dying) leprosy bacilli generate an increased activity of the immune system leading to acute inflammation of skin and nerves. These reactions can occur before MDT, but commence mostly during or after treatment. When not treated in time, the nerves can be severely damaged during these reactions i.e. neuropathy, which can 15

16 lead to disabilities. Leprosy is curable, but even when taking MDT the risk on neuropathy due to reactions is still present, until years after treatment has ended [34-36]. There are two types of reactions. Type 1 reactions, also called reversal reactions, arise in around 25-30% of the patients with borderline leprosy [37]. The number of skin lesions may increase and they become inflamed, red, swollen and tender. The peripheral nerves become swollen and the resulting intra-neural pressure may affect their function, leading to anaesthesia or paralysis of hands, feet and eyes [38]. Type 2 reactions, or erythema nodosum leprosum (ENL), only occur in lepromatous and borderline lepromatous types, respectively in approximately 10 and 50% of the patients [19,39]. ENL is seen as a chronic disease, since reactions can relapse over several years. Painful red papules or nodules appear on the skin. Next to involvement of nerves and skin, also other organs as testes, eyes, kidneys, joints and lymph nodes can be affected [40]. The reactions cause fever and general malaise and severe ENL can be life-threatening. Both type 1 and 2 reactions are treated with corticosteroids. The WHO advises a 12-week course of prednisolone, however, studies show that longer treatments may be more effective [13,41]. When patients with severe type 2 reactions do not respond well to prednisolone, thalidomide or clofazimine are given. iii. Nutrition and immunology As mentioned, nutritional status may influence the effectiveness of the immune response towards the leprosy bacilli or its antigens. The relation between nutrition, immunology and infections has been acknowledged for decades [42]. The essential defence against Mycobacterium leprae, an intracellular micro-organism, is dependent on the cell-mediated immune response [24]. Protein-energy malnutrition (PEM) as well as inadequate intake of vitamins and minerals are related to a reduced cell-mediated immunity [43-46]. For centuries (mal)nutrition has been linked to susceptibility to leprosy, for example by the Dutch physician Fortestu in the 17 th century [47]. Various foods as fish, salt, pork meat and alcoholic drinks have been blamed for causing leprosy [48]. However, until this day only little research has been carried out on the exact role of nutrition on the susceptibility and development of leprosy and thus this relation remains unclear. With the long incubation time of M. leprae it is very hard to study the nutritional status at the moment of infection. In addition, the relation between nutrition and infection runs both ways: infections may have major impact on nutritional status as well. This makes it almost impossible to distinguish between cause and consequence. 16

17 4. Leprosy neuropathy i. Neuropathy and nerve function impairment Leprosy is regarded as a disease of skin and nerves, but it is the neurological aspect that makes leprosy a disease with a high impact. Neuropathy can arise gradually or can be a consequence of reactions. New patients often present with some level of nerve function impairment (NFI), the percentage varies between 2% and 55%, with lower figures for PB cases [34,49-51]. On top of that, % of the patients develops neuropathy during or after treatment [35,50,52], also depending on classification and pre-existing nerve function impairments. M. leprae bacilli settle and reproduce in the Schwann cells [53,54]. The severity and extensiveness of neuropathy depends on the type of leprosy. Patients on the tuberculoid end of the spectrum often have only one or few nerves affected. Granulomatous inflammation causes enlargement of the nerves, oedema and loss of nerve tissue and structure. The damage occurs fast and severe. At the lepromatous end, often multiple nerves are bilaterally and symmetrically involved [55]. The lack of cell mediated immunity results in extensive infiltration of bacilli. The continued multiplication and high bacterial load cause damage to the nerves. This process is very slow, the nerve structure and function are often maintained for a long period of time. In borderline leprosy, the worst of the two above mentioned classes is combined: the wide involvement of nerves as in LL with the formation of granulomas and accompanying destruction of nerves as in TT. Patients in the borderline groups have therefore a high chance on severe neuropathy [53]. Generally in leprosy, sensory nerves are affected before motor nerves. Older histopathological studies showed that neuropathy occurs first in the small nerve fibres (unmyelinated C-fibres followed by myelinated Aδ-fibres), which conduct pain, warm and cold sensation and are responsible for the autonomic function (i.e. sweating) [56-58]. Subsequently, the large fibres (Aβ-fibres) are affected, responsible for vibration, touch and pressure perception. The INFIR study found, however, that the order of first affected modality and nerve fibre differs from patient to patient [59]. When autonomic nerves are affected, sweat gland function is lost, which leads to dryness of the skin and an increased risk of ulceration. The specific nerves affected by leprosy are the facial nerve, the ulnar, median and the radial cutaneous in the arm and hand, and the posterior tibial and the sural in the leg and foot. The most commonly affected nerves are the posterior tibial, sural and ulnar nerve [35,60,61]. To estimate which patients are at risk of developing NFI, Croft and colleagues developed the Clinical Prediction Rule. They established that MB patients who already have some nerve function impairment have the highest risk for new NFI and should have regular follow-ups in the first 2 years after diagnosis [62]. 17

18 ii. Disabilities When nerve function impairment is not treated within 6 months of onset, the damage can become irreversible and this will have severe consequences. Insensible feet and hands are extra vulnerable for burns and ulcers, with amputation as a result in the most extreme cases. This neurological aspect of leprosy, and the possibility of developing chronic disabilities, is what make leprosy such a serious disease. The disabilities make patients vulnerable for stigma and exclusion. Early detection of leprosy and neuropathy is therefore very important. iii. Diagnosis and nerve function assessment Diminished sweat function or blood flow (autonomic), pain, touch, pressure, warmth, cold and vibration sensation (sensory), and muscle strength (motor) can be assessed to detect leprosy neuropathy. Common methods used in the field to detect sensory nerve function impairments are monofilament testing (MFT) and ball point testing (BPT); both assess touch sensation on the hands and feet. A standard set of Semmes-Weinstein monofilaments consists of 6 filaments, with increasing thickness and pressure (from 70 mg to 300 mg). This is a reliable and standardized method to test for sensory impairments, in contrast to a BPT for which pressure is not standardized and which can only provide a yes/no outcome, although the low costs and high availability give BPT a huge advantage. For the detection of motor nerve function impairments, voluntary muscle testing (VMT) is performed by examining the ability of the patient to move a body part to a given position and to hold that position against resistance applied by the tester [63]. The modified 0 5 MRC scale is used, ranging from full range of motion and full resistance to complete paralysis. Both VMT and MFT require training and practice, but then both can be executed reliably [64-66]. When the nerve function is impaired according to one or both of these tests, it is seen as clinically impaired. The INFIR study showed that neuropathy was more widespread than could be detected with the MFT and VMT [67]. Before the nerve function impairment is clinically detectable, the majority of nerves already showed some subclinical neuropathy as can be detected with more sensitive methods. The warm detection threshold (WDT) and nerve conduction studies (NCS) were able to detect subclinical neuropathy up to 12 weeks before the clinical neuropathy was noticeable with MFT or VMT. Nerve conduction studies basically assess how fast an electrical pulse is conducted through a nerve. This can both be done for sensory and motor nerves, and the latency, amplitude and velocity of the pulse are recorded. The disadvantage of these two methods is that they are very expensive and require electricity and acclimatized conditions. When WDT and/or NCS are impaired, it is seen as subclinical impairment. 18

19 iv. Treatment of nerve function impairments Nerve function impairment of recent onset ( 6 months) can be treated with corticosteroids [35, 38]. The standard treatment recommended by the WHO starts at mg per day and is decreased over 12 weeks [68]. Ideally, each patient would receive an individual, tailored therapy, but this is only possible in referral centres. In the non-specialized clinics in the field, standard treatment is used. Several studies have been conducted to examine treatment of clinical NFI using prednisolone. In one cohort, the WHO recommended 12-week prednisolone regimen was found not to be successful in the prevention and reversal of NFI in MB patients [69]. Also a 16-week prednisolone regimen, usual practice nowadays, was found not to be very effective in two randomized controlled trials (RCT) [70,71]. There is reason to assume that longer steroid treatment might be more beneficial, since one RCT showed that a 5-month corticosteroid regimen was significantly more effective than a 3-month regimen. Since steroids can have severe side effects as diabetes, peptic ulcer, osteoporosis, glaucoma, cataract, psychosis, and hypertension, it is important that there is clear evidence for longer use of steroids. A Cochrane review concluded, however, that the evidence of the RCTs is not enough to draw robust conclusions about the long-term effect of corticosteroids on reactions and NFI in leprosy [53]. More research needs to be done to determine the optimal dose and duration of prednisolone for treating clinical NFI in leprosy. Interestingly, several studies have reported spontaneous recovery of nerve function impairments, even for old impairments. The Tripod studies showed that after 12 months 75% and 50% of the nerves spontaneously healed in mild and long-standing impairment, respectively. This was also seen in the AMFES trial in Ethiopia, where acute neuropathy recovered spontaneously in 42% and old neuropathy recovered in 23% of the cases [35,72]. 19

20 5. The TENLEP study Nerve function impairment is a vicious but frequent consequence of leprosy. Unfortunately, optimal treatments to restore and prevent NFI have not unequivocally been established yet. For this reason, the TENLEP study (Treatment of Early Neuropathy in LEProsy) was set up in four Asian countries: Bangladesh, India, Indonesia and Nepal. The study consists of two randomized controlled trials, one focusing on restoring and one on preventing NFI. The aim of the first trial was to assess whether prednisolone treatment of 32 weeks duration is more effective than treatment of 20 weeks duration in restoring nerve function in patients with clinical sensory and/or motor NFI of recent onset (<6 months). The second trial was designed to determine whether prednisolone treatment of early subclinical neuropathy, as detected with WDT and NCS, would prevent clinical sensory and/or motor function loss in leprosy patients. In both trials, patients were followed for 18 months. The first patient was taken in in June 2012 and the data collection was completed in October In addition to the primary aims of the study, the information gathered from this study may be useful to refine the clinical prediction rule as designed by Croft et al [73]. Guidelines for prophylactic prednisolone could be developed for certain high risk groups to make steps forward in the prevention of disabilities and deformities in newly diagnosed leprosy patients. However, this is outside the scope of this thesis. The study took place in six leprosy centres: Anandaban Leprosy Hospital and Lalgadh Leprosy Services Centre in Nepal, the Foundation for Medical Research and the JALMA institute in India, Dr. Soetomo hospital in Surabaya, Indonesia, and Nilphamari-Leprosy Mission Hospital in Bangladesh (see maps in 2.ii). 20

21 6. Research questions The first objective of this thesis is to study methods that help early detection and treatment of neuropathy in order to prevent irreversible nerve function impairment and the resulting disabilities in leprosy patients. Regarding risk factors for leprosy we studied differences in diet and food security between leprosy patients and a control group without leprosy. The research questions of this thesis are: 1. How can the detection of nerve function impairment in leprosy at field level be improved? 2. How can irreversible nerve function impairment best be prevented in leprosy? 3. Which nutritional factors possibly underlie the development of clinical leprosy? This thesis is divided in three sections. In the first part, the focus lays on methodological studies. The TENLEP study protocol is presented in Chapter 2, describing the details of the assessments and treatment for both the Clinical and Subclinical trial in depth. Chapters 3, 4 and 5 cover the first research question. In Chapter 3, a study in India and Nepal defining the normal values for monofilament tests for two additional nerves is described. The results of reliability testing for monofilament and voluntary muscle testing in the six TENLEP centres are given in Chapter 4. Chapter 5 presents a study in which several new portable, battery operated tests are compared to determine if they are useful in early detection of leprosy neuropathy in the field. The second section of this thesis concentrates on study results. Chapter 6 answers the second research question by presenting the results of the clinical TENLEP trial, aimed to examine whether prednisolone can restore clinical nerve function impairment in leprosy patients. The serious adverse events in the TENLEP trials are addressed in Chapter 7. Finally, Chapter 8 discusses the results of a case-control study in Bangladesh, comparing the diet and food security between newly diagnosed leprosy patients and a control population. In the third and final part, Chapter 9, the results are discussed and the research questions are answered. 21

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23 16. World Health Organization, Leprosy Elimination Project: status report , WHO: Geneva. 17. Meima, A., J.H. Richardus, and J.D.F. Habbema, Trends in leprosy case detection worldwide since Leprosy Review, (1): p World Health Organization, Global leprosy update 2014: need for early case detection, in Weekly Epidemiological record. 2015: Geneve. p Britton, W.J. and D.N.J. Lockwood, Leprosy. Lancet, (9416): p Thorat, D.M. and P. Sharma, Epidemiology, in IAL textbook of leprosy, H.K. Kar and B. Kumar, Editors. 2010, Jaypee Brothers Medical Publishers: New Delhi. 21. Montestruc, E. and R. Berdonneau, 2 New cases of leprosy in infants in Martinique. Bulletin de la Société de pathologie exotique et de ses filiales, (6): p Ridley, D.S. and W.H. Jopling, Classification of leprosy according to immunity. A fivegroup system. Int J Lepr Other Mycobact Dis., (3): p Montoya, D. and R.L. Modlin, Learning from leprosy: insight into the human innate immune response. Adv Immunol, : p Harboe, M., The immunology of leprosy, in Leprosy, C. Hastings, Editor. 1985, Churchill Livingstone: Edinburgh. 25. Mishra, R.S. and J. Kumar, Classification, in IAL textbook of leprosy, H.K. Kar and B. Kumar, Editors. 2010, Jaypee Brothers Medical Publishers: New Delhi. 26. Izumi, S., et al., An epidemiological study on Mycobacterium leprae infection and prevalence of leprosy in endemic villages by molecular biological technique. Indian J Lepr., (1): p Klatser, P.R., et al., Detection of Mycobacterium leprae nasal carriers in populations for which leprosy is endemic. J Clin Microbiol., (11): p Lavania, M., et al., Cohort study of the seasonal effect on nasal carriage and the presence of Mycobacterium leprae in an endemic area in the general population. Clinical Microbiology and Infection, (10): p Godal, T., M. Lofgren, and K. Negassi, Immune response to M. leprae of healthy leprosy contacts. Int J Lepr Other Mycobact Dis., (3): p Moet, F.J., et al., Effectiveness of single dose rifampicin in preventing leprosy in close contacts of patients with newly diagnosed leprosy: cluster randomised controlled trial. BMJ, (7647): p Scollard, D.M., et al., The continuing challenges of leprosy. Clin Microbiol Rev, (2): p Gaschignard, J., et al., Pauci- and Multibacillary Leprosy: Two Distinct, Genetically Neglected Diseases. PLoS Negl Trop Dis, (5): p. e

24 33. Sauer, M.E., et al., Genetics of leprosy: expected and unexpected developments and perspectives. Clin Dermatol, (1): p Croft, R.P., et al., Incidence rates of acute nerve function impairment in leprosy: A prospective cohort analysis after 24 months (The Bangladesh Acute Nerve Damage Study). Leprosy Review, (1): p Saunderson, P., et al., The pattern of leprosy-related neuropathy in the AMFES patients in Ethiopia: Definitions, incidence, risk factors and outcome. Leprosy Review, (3): p Richardus, J.H., et al., Nerve function impairment in leprosy at diagnosis and at completion of MDT: A retrospective cohort study of 786 patients in Bangladesh. Leprosy Review, (4): p van Brakel, W.H., I.B. Khawas, and S.B. Lucas, Reactions in leprosy: An epidemiological study of 386 patients in West Nepal. Leprosy Review, (3): p Britton, W.J., The management of leprosy reversal reactions. Leprosy Review, (3): p Pocaterra, L., et al., Clinical course of erythema nodosum leprosum: an 11-year cohort study in Hyderabad, India. Am J Trop Med Hyg., (5): p Pfaltzgraff, R.E. and A. Bryceson, Clinical leprosy, in Leprosy, C. Hastings, Editor. 1985, Churchill Livingstone: Edinburgh. 41. Sundar Rao, P.S.S., et al., Multi-centre, double blind, randomized trial of three steroid regimens in the treatment of type-1 reactions in leprosy. Leprosy Review, (1): p Scrimshaw, N.S., C.E. Taylor, and J.E. Gordon, Interactions of nutrition and infection. The American journal of the medical sciences, (3): p Ferguson, A. and G.E. Griffin, Nutrition and the immune system, in Human Nutrition and Dietetics, J.S. Garrow, W.P.T. James, and A. Ralph, Editors. 2000, Elsevier Science Ltd.: London. p Keusch, G.T., The history of nutrition: malnutrition, infection and immunity. J Nutr., (1): p. 336S-340S. 45. Scrimshaw, N.S. and J.P. SanGiovanni, Synergism of nutrition, infection, and immunity: an overview. Am J Clin Nutr., (2): p. 464S-477S. 46. Edelman, R., Malnutrition and leprosy--an analytical review. Lepr India, (3): p Keil, E.G., The importance of nutrition in the prevention and cure of leprosy. International Journal of Leprosy, (4): p Klingmüller, V., Nahrung als Ursache von Lepra, in Die Lepra. 1930, Springer-Verlag: Berlin. p

25 49. van Brakel, W.H., Peripheral neuropathy in leprosy and its consequences. Lepr Rev., (Suppl): p. S van Brakel, W.H., et al., The INFIR Cohort Study: investigating prediction, detection and pathogenesis of neuropathy and reactions in leprosy. Methods and baseline results of a cohort of multibacillary leprosy patients in north India. Lepr Rev, (1): p Jiang, J., et al., A field trial of detection and treatment of nerve function impairment in leprosy - Report from national POD pilot project. Leprosy Review, (4): p Smith, W.C., et al., Predicting neuropathy and reactions in leprosy at diagnosis and before incident events-results from the INFIR cohort study. PLoS Negl Trop Dis, (8): p. e Ridley, D.S. and C.K. Job, The pathology of leprosy, in Leprosy, C. Hastings, Editor. 1985, Churchill Livingstone: Edinburgh. 54. Rao, P.N. and S.K. Suneetha, Neuritis: Definition, Clinical manifistations and Proforma to Record Nerve Impairment, in IAL textbook of leprosy, H.K. Kar and B. Kumar, Editors. 2010, Jaypee Brothers Medical Publishers: New Delhi. 55. Agrawal, A., et al., Neurological manifestations of Hansen's disease and their management. Clinical Neurology and Neurosurgery, (6): p Antia, N.H., The significance of nerve involvement in leprosy. Plast Reconstr Surg, (1): p Shetty, V.P., N.H. Antia, and J.M. Jacobs, The pathology of early leprous neuropathy. Journal of the Neurological Sciences, (1-3): p Shetty, V.P., et al., Study of evolution of nerve damage in leprosy. Part II--Observations on the index branch of the radial cutaneous nerve in contacts of leprosy. Lepr India, (1): p McKnight, J., Clinical testing and prognostic markers for the development of leprosy neuropathy, in Infectious and Tropical Diseases. 2010, University of London: London. 60. Croft, R.P., et al., Nerve function impairment in leprosy: design, methodology, and intake status of a prospective cohort study of 2664 new leprosy cases in Bangladesh (The Bangladesh Acute Nerve Damage Study). Lepr Rev, (2): p van Brakel, W.H., et al., The INFIR Cohort Study: assessment of sensory and motor neuropathy in leprosy at baseline. Lepr Rev, (4): p Croft, R.P., et al., A clinical prediction rule for nerve function impairment in leprosy patients-revisited after 5 years of follow-up. Lepr Rev, (1): p Brandsma, J.W. and W.H. van Brakel, Protocol for motor function assessment in leprosy and related research questions. Indian J Lepr, (2): p

26 64. Anderson, A.M. and R.P. Croft, Reliability of Semmes Weinstein monofilament and ballpoint sensory testing, and voluntary muscle testing in Bangladesh. Leprosy Review, (3): p Brandsma, J.W., et al., Intertester reliability of manual muscle strength testing in leprosy patients. Lepr Rev, (3): p Roberts, A.E., et al., Ensuring inter-tester reliability of voluntary muscle and monofilament sensory testing in the INFIR Cohort Study. Lepr Rev, (2): p van Brakel, W.H., et al., Early diagnosis of neuropathy in leprosy--comparing diagnostic tests in a large prospective study (the INFIR cohort study). PLoS Negl Trop Dis, (4): p. e World Health Organization. WHO Model Prescribing Information: Drugs Used in Leprosy [cited 2015 May 7th]; Available from: Shetty, V.P., et al., The effect of corticosteroids usage on bacterial killing, clearance and nerve damage in leprosy; part 3 - study of two comparable groups of 100 multibacillary (MB) patients each, treated with MDT 1 steroids vs MDT alone, assessed at 6 months post - release from 12 months MDT. Leprosy Review, (1): p van Brakel, W.H., et al., The prognostic importance of detecting mild sensory impairment in leprosy: A randomized controlled trial (TRIPOD 2). Leprosy Review, (4): p Walker, S.L., et al., A phase two randomised controlled double blind trial of high dose intravenous Methylprednisolone and oral prednisolone versus intravenous normal saline and oral prednisolone in individuals with leprosy type 1 reactions and/or nerve function impairment. PLoS Neglected Tropical Diseases, (4). 72. Croft, R.P., et al., The treatment of acute nerve function impairment in leprosy: results from a prospective cohort study in Bangladesh. Lepr Rev., (2): p Croft, R.P., et al., A clinical prediction rule for nerve-function impairment in leprosy patients. Lancet, (9215): p

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29 Chapter 2 Two randomized controlled clinical trials to study the effectiveness of prednisolone treatment in preventing and restoring clinical nerve function loss in leprosy: the TENLEP study protocols Inge Wagenaar Wim Brandsma Erik Post Wim van Brakel Diana Lockwood Peter Nicholls Paul Saunderson Cairns Smith Einar Wilder-Smith Jan Hendrik Richardus BMC Neurology 2012, 12:159

30 Abstract Introduction Nerve damage in leprosy often causes disabilities and deformities. Prednisolone is used to treat nerve function impairment (NFI). However, optimal dose and duration of prednisolone treatment has not been established yet. Besides treating existing NFI it would be desirable to prevent NFI. Studies show that before NFI is clinically detectable, nerves often show subclinical damage. Within the Treatment of Early Neuropathy in LEProsy (TENLEP) study two double blind randomized controlled trials (RCT) will be carried out: a trial to establish whether prednisolone treatment of 32 weeks duration is more effective than 20 weeks in restoring nerve function in leprosy patients with clinical NFI (Clinical trial) and a trial to determine whether prednisolone treatment of early subclinical NFI can prevent clinical NFI (Subclinical trial). Methods Two RCTs with a follow up of 18 months will be conducted in six centres in Asia. In the Clinical trial leprosy patients with recent (<6 months) clinical NFI, as determined by Monofilament Test and Voluntary Muscle Test, are included. The primary outcomes are the proportion of patients with restored or improved nerve function. In the Subclinical trial leprosy patients with subclinical neuropathy, as determined by Nerve Conduction Studies (NCS) and/or Warm Detection Threshold (WDT), and without any clinical signs of NFI are randomly allocated to a placebo group or treatment group receiving 20 weeks prednisolone. The primary outcome is the proportion of patients developing clinical NFI. Reliability and normative studies are carried out before the start of the trial. Discussion This study is the first RCT testing a prednisolone regimen with a duration longer than 24 weeks. Also it is the first RCT assessing the effect of prednisolone in the prevention of clinical NFI in patients with established subclinical neuropathy. The TENLEP study will add to the current understanding of neuropathy due to leprosy and provide insight in the effectiveness of prednisolone on the prevention and recovery of NFI in leprosy patients. In this paper we present the research protocols for both Clinical and Subclinical trials and discuss the possible findings and implications. 30

31 Introduction Damage to peripheral nerves is the main consequence of leprosy and may cause deformities and disabilities in patients. Nerve damage can occur before, during and after multidrug therapy (MDT) and is a result of inflammation in the nerves due to immunological reactions [1]. For many years corticosteroids, mostly prednisolone, have been used to treat nerve function impairment (NFI) in leprosy patients [2]. However, an optimal dose and duration of steroid treatment has yet to be established [3]. In addition, research should focus also on possibilities of timely detection and treatment of early (subclinical) neuropathy in order to prevent NFI and its consequences [4]. The TENLEP study is designed to obtain additional information about prednisolone treatment for preventing and restoring nerve function in people affected by leprosy. Within the TENLEP study two randomized clinical trials will be conducted; one trial focusing on patients with subclinical neuropathy, and the second trial focusing on patients with clinical NFI. Between 6 and 27% of the newly detected leprosy cases in 2010 [5] presented with visible impairment (grade 2 disability) [2,4]. This WHO leprosy disability grading system is the most widely used method to assess impairment in leprosy patients and is generally used for monitoring program quality [6,7]. More accurate assessments for NFI are Voluntary Muscle Testing (VMT) and Monofilament Testing (MFT) or ball point tests [8], which are widely used in clinical practice to assess motor and sensory NFI, respectively. Recently, more sensitive methods have been introduced to detect early, subclinical neuropathy. The INFIR study found that nerve conduction studies (NCS) and Warm Detection Threshold (WDT) are the most effective methods for finding subclinical nerve damage [1]. With these tests subclinical neuropathy can be detected at least 3 months before VMT and MFT can determine the first clinical impairments. Subclinical changes during and following MDT were also found to be predictive of new onset NFI [9]. To examine treatment of clinical NFI several studies have been conducted using prednisolone. In one cohort the WHO recommended prednisolone regimen (starting with 40 mg prednisolone/day, tapered down over 12 weeks [2]) was found not to be successful in the prevention and reversal of NFI in multibacillary (MB) patients treated for reactions or neuropathy [10]. Also a 16-week prednisolone regimen, usual practice nowadays, was found not to be very effective in two randomized controlled trials (RCT) [11,12]. One trial was in patients with Type 1 Reactions and/or NFI, receiving either prednisolone or prednisolone with intravenous methylprednisolone. Close to 50% of the patients required additional prednisolone during or after the treatment period [11]. The second RCT in MB patients with mild sensory impairment did not show a difference in NFI as measured with MFT 12 months after start of prednisolone treatment [12]. Van Veen et al. [3], reviewing RCTs comparing placebo to prednisolone treatment, conclude that studies so far have not provided enough evidence to draw robust conclusions about the long-term effect of corticosteroids on reactions and NFI. However, there is reason to assume that longer steroid treatment might be more beneficial, since one study showed that a 5-month corticosteroid regimen was significantly more effective than a 3-month 31

32 regimen [13]. In TENLEP we aim to assess whether prednisolone treatment of 32 weeks duration is more effective than treatment of 20 weeks duration in restoring nerve function in patients with clinical sensory and/or motor NFI of recent onset (<6 months), as detected by VMT and/or MFT. The effect of prednisolone treatment on patients with subclinical neuropathy to prevent clinical NFI as determined with MFT and/or VMT has not been studied in a RCT before. The objective of the Subclinical trial is therefore to determine whether prednisolone treatment of early subclinical neuropathy, as detected with WDT and NCS, would prevent clinical sensory and/or motor function loss in leprosy patients. This paper presents the protocols for both the Clinical and the Subclinical trial. Methods Definitions General Neuropathy (peripheral) Functional impairment and/or structural damage to autonomic, sensory, and motor nerve fibres within the peripheral nervous system. Nerve function impairment Sensory, motor or autonomic neuropathy evidenced by clinically detectable reduction in function in sensory, motor and/or autonomic fibres. The level of impairment that is clinically detectable depends on the sensitivity of the testing instruments used. (It does not include abnormality of nerve conduction that is detectable only by electrophysiological means and WDT). Nerve damage An imprecise but common term for neuropathy, which is also used in relation to trauma. Here it indicates clinical or subclinical damage to a nerve, whether reversible or irreversible. Neuritis A condition in which inflammatory cells are found in the nerve, detectable by swelling and/or functional impairment with spontaneous nerve pain and/or nerve tenderness on palpation. 32

33 Subclinical neuropathy Patients have normal values for Voluntary Muscle Testing and Monofilament Testing, but are impaired on Nerve Conduction Studies (NCS) and/or Warm Detection Threshold (WDT). Entry criteria Clinical trial At least one nerve with either a VMT score of 4 or less on the 0 5 (modified) MRC scale or with a monofilament threshold increased compared to normal subjects by three or more monofilament levels on any test-site, two levels on one test-site AND at least one level on another test-site, OR one level on three or more test-sites for one nerve. With VMT each nerve is tested with one specific test assessing the strength of a muscle (group) innervated by that nerve. For MFT each nerve is tested on three sites. Entry criteria Subclinical trial Any one parameter (Motor Nerve Conduction, Sensory Nerve Conduction, Warm Detection Threshold) abnormal in at least two nerves or any two parameters abnormal in at least one nerve. VMT and MFT values are normal. Outcome criteria Clinical trial Restored nerve function Monofilament Test and/or Voluntary Muscle Test of a nerve are recovered to normal levels (MFT = 0, VMT = 5). Improved nerve function At least one nerve shows better results on Monofilament Test and/or Voluntary Muscle Test. The MF thresholds should be reduced by three or more monofilament levels on any site, two levels on one site AND at least one level on another site, OR one level on three or more sites for one nerve. VMT score should be increased by at least 1 point. Improved Reaction Severity Scale score When the score on the Reaction Severity Scale [14] decreases by at least 3 points on the sum score or at least 2 points on any individual item in the scale. Count of nerve function impairments (CNFI) The sum of MFT and VMT scores (5 being normal) for all nerves tested in the study. 33

34 Improved SALSA scale score The SALSA scale [15] score decreases at least with 1 category in standardized categories of SALSA values as described in SALSA Scale User s Manual (Salsa Scale User s Manual, Version 1.1, July 2010) Improved Participation scale score The Participation scale [16] score decreases at least 1 grade on the Grades of participation restriction scale (as described on P-scale form). Outcome criteria Subclinical trial Clinical motor impairment (MI) Motor neuropathy resulting in weakness of the muscles innervated by a given nerve. A patient is diagnosed as having clinical motor impairment if the VMT score for a muscle test is 4 or less on the 0 5 (modified) MRC scale. If a score of 4 is found, the test will be repeated by a second assessor. Clinical sensory impairment (SI) A patient is diagnosed as having sensory impairment of a nerve if the monofilament threshold is increased by three or more monofilament levels on any site, two levels on one site AND at least one level on another site, OR one level on three or more sites for one nerve. Subclinical nerve function score (SNFS) The Subclinical Nerve Function Score (SNFS) will be computed for NC parameters (amplitude and latency) and warm detection threshold, where non-impaired parameter adds 1 point to the overall score. Improved SNFS Subclinical Nerve Function Score increased with at least 1 point Deteriorated SNFS Subclinical Nerve Function Score decreased with at least 1 point Design of study The TENLEP study consists of two multi-centre randomized triple blind controlled trials, both with two treatment arms, to study the effectiveness of prednisolone treatment restoring (Clinical trial) and in preventing (Subclinical trial) clinical nerve function loss. 34

35 Six institutions in four different countries participate in this study: Nepal (Lalgadh Hospital and Anandaban Hospital); India (JALMA Institute for Leprosy -Agra and Foundation for Medical Research-Mumbai); Bangladesh (Nilphamari Hospital); and Indonesia (Dr. Soetomo Hospital- Surabaya). Anandaban Hospital and Dr Soetomo Hospital will only take part in the Clinical trial. All collaborative institutions are referral hospitals specialized in the detection and treatment of leprosy. At each institution a Principal Investigator (PI) is responsible for the research at that centre. The overall responsibility lies with the International Coordinator (dr. E. Post), guided by an International Steering Committee. Participants In the Clinical trial leprosy patients with clinical NFI will be randomly allocated to either treatment of standard duration (20 weeks) or an interventional treatment of longer duration (32 weeks). Both multibacillary (MB) and paucibacillary (PB) patients diagnosed with clinical sensory and/or motor nerve impairment of less than six months duration are enrolled. For the Subclinical trial leprosy patients with subclinical neuropathy will be randomly divided into an intervention group and a placebo group. Newly registered MB and PB patients without clinical NFI but having subclinical sensory and/or motor neuropathy at diagnosis, or developing this within their first three months of MDT treatment, will be eligible for inclusion in the trial. Patients from the Subclinical trial developing NFI in the first three months of the trial can enter the Clinical trial, but only data of patients that were allocated to the placebo group of the Subclinical trial will be analysed. Inclusion and exclusion criteria Patients included in the trials should be between 15 and 60 years of age, must give informed consent and should be free from conditions that may affect the peripheral nervous system, such as diabetes mellitus, and other active underlying diseases for example hypertension, osteoporosis and tuberculosis. Included patients receive deworming treatment with Mebendazol before steroid treatment starts. In both studies patients will be followed-up for 18 months. Excluded will be women pregnant at diagnosis, patients who need steroids for reasons other than recent NFI and patients with a single skin lesion on the trunk as the only sign of leprosy. Randomization and blinding In both trials patients will be randomly allocated to one of the two study arms. Randomization tables are provided by the statistician and drugs are labelled accordingly by the manufacturer. The key is held by the International Coordinator, Study Manager and PI of each centre and will be broken after the data analysis is completed or earlier for patients with adverse events, and new or worsening NFI. 35

36 Sample size calculation For the Clinical trial a recovery of 60% is presumed for the standard regimen in the control group [17]. To detect a recovery of 70% in the treatment group, compensating for 20% of loss to follow-up, a total of 720 subjects need to be enrolled. The INFIR study shows that of the leprosy patients having subclinical neuropathy 16% will eventually develop clinical NFI [1]. For the Subclinical trial we assume a reduction in patients developing clinical NFI by half in the treated group (8%). Anticipating a loss to follow-up of 20% at 18 months, a sample size of 275 subjects per trial arm is required. For both sample size calculations, a one-tailed alternative hypothesis, 80% power and 5% significance is used Follow-up Patients will be followed-up for 18 months. Nerve function is monitored by monthly VMT and MFT in both trials and assessments with NCS and WDT takes place at 20 weeks, 12 and 18 months from intake in the Subclinical trial. Participants can be withdrawn from the trials because of medical or clinical reasons or pregnancy. Subjects missing their appointment will be visited at home by research staff within two weeks when contact by cell phone is not possible or did not result in late attendance at the clinic. In cases when only one appointment is missed and medication is not taken for a maximum of one month, treatment will continue from the first package missed. The TENLEP trials started in April 2012, with a planned duration of intake of 1.5 years. Medication In both trials the medication provided is prednisolone. Prednisolone will be allocated based on two bodyweight classes. The low weight class (patients below 50 kg), will receive prednisolone treatment based on a body weight of 45 kg. The high weight class (patients 50 kg and more) receives treatment based on a body weight of 60 kg. Treatment arms In the Clinical trial patients in the intervention arm receive prednisolone for 32 weeks, in tablets of 5 mg. The control arm will follow a regimen of 20 weeks and receive placebo tablets to keep the number of tablets equal to the treatment group for effective treatment time and duration. The dose in both treatment and control arm starts at 1 mg/kg/day (either 45 or 60 mg/day depending on weight class) and will be tapered down over 32 and 20 weeks, respectively. Figure 1.A shows the dose over time of both arms in the Clinical trial. In contrast to previous trials, the middle range of the prednisolone dose will be maintained at a high level (0.44 mg/kg/day) for a longer period (12 weeks). In the Subclinical trial patients receive either prednisolone or placebo for 20 weeks in tablets of 5 mg. The prednisolone dose starts at 1 mg/kg/day (either 45 or 60 mg/day depending on weight class) and will be tapered down over 20 weeks. Figure 1.B shows the timeline and dosage. The total dosage of prednisolone over 20 36

37 weeks will be 2.8 grams for patients under 50 kg body weight, and 3.7 grams for patients over 50 kg body weight. 1.2 Prednisolone dose (mg/kg/day) week arm 32-week arm Week A Prednisolone dose (mg/kg/day) Prednisolone Group Week B Figure 1 A- Timeline prednisolone dose for patients under 50 kg in the Clinical trial. Figure 1 B- Timeline prednisolone dose for patients under 50 kg in the Subclinical trial 37

38 Adverse events Prednisolone has known adverse events, ranging from mild adverse events, such as moon face, fungal infections and acne, to serious adverse events such as peptic ulcer, osteoporosis glaucoma, cataract, psychosis, diabetes and hypertension [18,19]. Serious adverse events have not been encountered frequently in previous trials studying prednisolone for treating NFI in leprosy patients [20], however strict monitoring of adverse events will take place in the TENLEP trials. In case of serious adverse events the PI will break the key and initiate an individualized treatment scheme. In the event of minor side effects additional medication will be prescribed according to normal protocol in the clinic, but the key will not be broken. Data collection General assessments Before intake, the general health of all possible eligible patients will be checked and history will be taken according to the protocol. Additionally, patients will be tested for specific medical conditions related to neuropathy and prednisolone intake such as diabetes mellitus (urine test) and osteoporosis (FRAX). Leprosy status will be categorized using both WHO classification (PB/MB), and Ridley-Jopling classification [21]. The latter will be done on clinical grounds, but a skin biopsy can be taken on voluntary basis for confirmation of the classification by a trained pathologist. For this procedure an additional consent procedure is in place. Of each patient a slit skin smear will be taken for determining the Bacteriological Index (BI). Clinical nerve function tests After intake, eligible subjects will be assessed for clinical sensory and motor nerve impairment. The clinical sensory function is tested with monofilaments using a standard set of 5 Semmes- Weinstein monofilaments ranging from blue (200 mg) to pink (300 g) [22]. This test of touch sensibility is based on indenting the skin surface with a series of standard nylon filaments. For each thickness it is recorded whether or not the patient feels the touch, starting with the thinnest filament. For hands the normal threshold is the blue filament and for feet the purple (2 mg) filament; when these filaments are felt by the patient it results in a score of 0. The score increases with 1 for each thicker filament not felt, with a maximum score of 5 in hands and 4 in feet (filament of 300 g not felt) for every site. Each nerve will be tested on 3 sites. The trigeminal nerve is tested by assessing the blink regularity. A patient is diagnosed as having sensory impairment if the monofilament threshold is increased by three or more monofilament levels in one single nerve (over 3 sites). Motor nerve impairment will be assessed with Voluntary Muscle Testing (VMT) using the 0 5 MRC scale [23,24]. The test is performed by checking the ability of the patient to move a body part to a given position and to hold that position against resistance applied by the tester. A nerve scoring lower than 5 is considered impaired. When a single score of 4 is found this score should be independently confirmed by a second assessor. 38

39 Subclinical nerve function tests When new patients show no abnormal values by VMT and MFT they will be tested further for possible inclusion in the Subclinical trial. For detecting subclinical neuropathy sensory and motor Nerve Conduction Studies (NCS), carried out with the Neurocare 2000 EMG machine (BioTech Ltd, Mumbai), and Warm Detection Threshold (WDT) test, using the TSA II (MEDOC, Israel), will be performed. NCS and WDT will take place in an air-conditioned room at approximately C. All assessments will be carried out at both sides of the body. Table 1 shows which nerves will be tested and methods used. MFT and VMT will be assessed at start of the trial and subsequently every month in both studies and follow-up periods on all subjects. WDT, Sensory Nerve Conduction (SNC) and Motor Nerve Conduction (MNC) will be carried out at baseline, end of treatment period (20 weeks) and at 12 and 18 months. For the Clinical trial additional information will be obtained using the Reaction Severity Scale (RSS) [25], the Screening of Activity Limitation and Safety Awareness (SALSA) scale [26] and the Participation Scale [27]. All scales will be filled in at baseline, end of treatment period (32 weeks), 12 and 18 months. Table 1- Nerves assessed by the different methods Nerve VMT MFT MNC SNC WDT Trigeminal blink Facial x Ulnar x x x x x Median x x x x x Radial x x x x Common peroneal x x Posterior tibial x x x x Sural x x x Reliability and normative studies Prior to intake inter-tester reliability studies have taken place for all assessments (MFT, VMT, WDT, NCS) to test and improve comparability of the results and ensure high measurement quality. After reliability studies for NCS and WDT, normative studies were carried out to establish the local normal values for each separate collaborative centre. For each nerve a minimum of 150 normal subjects from surrounding areas were tested, equally spread over both sexes and 3 age categories (15 30, and years of age). Normal subjects will be screened to ascertain they do not have diabetes or nerve function impairment. 39

40 Standard operating procedures and training For all assessments standard operating procedures (SOP) have been developed. To achieve consistency of the assessments between all research centres, the PI and two main assessors of each centre received training in research protocol procedures and handling equipment. In addition, an online Good Clinical Practice (GCP) course was completed by all PI s. Outcome measures The Clinical trial The primary study outcome is the proportion of patients with restored and improved nerve function (of all nerves) measured by VMT/MFT at 18 months. Secondary outcomes are based on results of the Reaction Severity Scale, SALSA and Participation Scale (Figure 2). Furthermore, a Count of Nerve Function Impairment (CNFI) will be computed to measure the results of treatment on nerve function, with special interest for most commonly affected nerves. The CNFI will consist of the count of nerve function impairments detected by monofilament and VMT testing and will be validated in this trial. The Subclinical trial The proportion of patients developing clinical NFI as measured by MFT and VMT is the primary outcome indicator (Figure 2). Secondary outcome measures focus on results of the specific assessments for detection of subclinical neuropathy (WDT, SNC and MNC). Data analysis After the data is entered for each centre, data will be checked and cleaned. Regular weekly back-ups will be made for local off-site storage in addition to a monthly upload of data via a web-based repository to the project statistician. Reliability studies Inter-rater reliability for categorical outcomes of VMT and MFT are assessed using weighted Kappa statistics. Comparing differences and averages of paired assessments for WDT, SNC and MNC tests are based on Bland-Altman plots. In addition, a mixed model analysis of variance will be used to compute the intraclass correlation coefficient. Normative studies To determine normative values for WDT and NCS for each centre, outliers are excluded before limits of normal function are calculated using an approach based on regression analysis taking into account age-related trends. The 97.5 th percentile is used to define abnormal function. 40

41 Primary outcomes Analysis of primary outcomes will be done at 20 (Subclinical trial) or 32 weeks (Clinical trial), 12 and 18 months. Possible effects of potential covariates (gender, age, leprosy classification) will be assessed with ANCOVA. All analyses will be carried out using Stata statistical software. Secondary outcomes Continuous outcome measures will be analysed with Analysis of (co)variance, assessing one or more covariates. Categorical outcomes may be analysed using chi-squared test or log linear models. For Survival outcomes the log rank test will be used. Ethics All national Research Ethics Committees have approved the study protocol, which are for India: Indian Council of Medical Research; Nepal: Nepal Health Research Council (NHRC); Indonesia: Komite Etik Penelitian Kesehatan RSUD Dr. Soetomo Surabaya; Bangladesh: Bangladesh Medical Research Council- National Research Ethics Committee. In addition, all local Research Ethics Committees have given their approval as well. Written consent will be obtained from individual subjects before inclusion and for minors additional consent from their guardians will be sought. Discussion This paper describes the protocols of two randomized controlled clinical trials within the TENLEP study. In the Clinical trial the effect of long term prednisolone treatment on the restoration of clinical NFI will be investigated. In the Subclinical trial the efficacy of prednisolone in preventing the development of clinical NFI, in patients with subclinical neuropathy, will be examined. The optimal dose and duration of prednisolone for treating clinical NFI in leprosy has not been established yet and there is not enough evidence available from randomized controlled clinical trials on the long term effect of prednisolone treatment [3]. The TENLEP study addresses this knowledge gap and will extend the current understanding of prednisolone regimens for the treatment of clinical NFI by comparing a prednisolone treatment of 32 weeks and 20 weeks under controlled circumstances. If a 32 weeks treatment proves to be effective in restoring or improving clinical NFI in leprosy patients, new guidelines can be developed which can significantly improve patient management in leprosy care and especially a positive effect on the prevention of disabilities (POD) can be expected. 41

42 Primary outcome: 1. Proportion of patients developing clinical NFI as defined by MFT/VMT change Eligible for initial tests All tests: neg VMT: neg MFT: neg WDT/NCV: pos 6 months: VMT/ MFT: pos > 6 months: VMT/ MFT: pos Reaction skin only (RR or ENL) Repeat tests at 1-3 months Subclinical trial Clinical trial Excluded Excluded, but steroids Prednisolone (20 weeks) Placebo (20 weeks) Prednisolone (32 weeks) Prednisolone (20 weeks) Repeat tests (20 weeks, 12 and 18 months) VMT/MFT monthly during treatment period Repeat tests (32 weeks, 12 and 18 months) VMT/MFT monthly during treatment period Secondary outcomes - detected by SC methods (WDT or NCS) 2. Proportion of patients with fully recovered SC function (all nerves) 3. Proportion of patients with improved, unchanged or deteriorated SC function (based on count of impaired functions) 4. Proportion of patients with recovered, improved, unchanged or deteriorated SC function of a given nerve 5. Spontaneous recovery of SC function (placebo group) 6. Proportions of improved, unchanged or deteriorated SC functions (Placebo group) 7. Proportion of patients with serious adverse events or other complications leaving the trial incl. skin reactions and ENL Primary outcome 1. Proportion of patients with restored or improved nerve function as measured by MFT/VMT (all nerves) Secondary outcomes (detected by VMT / MFT) 2. Proportion of patients with recovered improved, unchanged or deteriorated function of a given nerve (e.g. ulnar nerve) 3. Proportion with improved Count of Nerve Function Impairments (CNFI) Secondary outcome other: 4. Proportion of patients with improved Reaction Severity Scale 5. Proportion of patients with serious adverse events or other complications leaving the trial 6. Proportion of patients with improved SALSA & P-scale scores Figure 2- Overview of intake, assessments and outcomes in the Clinical and Subclinical trial

43 This study will be the first to evaluate prednisolone treatment for the prevention of clinical NFI in people affected by leprosy diagnosed with subclinical neuropathy. The results of the preceding TRIPOD trial provide some insight in possible effects of prednisolone in preventing new NFI [28]. To prevent new NFI and reactions, leprosy patients with and without pre-existing NFI at diagnosis (as determined with VMT and MFT) received a prophylactic low dose of prednisolone (20 mg/day) for four months, tapered down in the last month. Although a reduced incidence of new NFI and reactions was observed at the end of treatment at four months, this was not sustained at one year. More important however, the preventive effect of prednisolone at four months was more than three times higher in patients with no pre-existing NFI [28]. A second study that has some similarities with our Subclinical trial is of that of Capadia et al. (2010), who studied the effect of prednisolone on neuropathy as assessed with NCS. In their study, neuropathy was divided in mild, moderate and severe groups, as percentages deviating from normative values. Their findings suggest that a 12-week prednisolone course is not effective in preventing or reversing nerve damage. However, they found that mildly affected nerves showed higher improvement rates than moderately and severely affected nerves (53%, 21% and 14% of the nerves improved respectively). The better outcomes of prednisolone treatment and prophylaxis on non-affected and mildly affected nerves from both studies of TRIPOD and Capadia et al. [29] show the importance of early treatment of neuropathy in leprosy patients. With the Subclinical trial we hope to establish that prednisolone treatment can prevent the development of clinical NFI and in this way can prevent disabilities and deformities in newly diagnosed leprosy patients. If the prednisolone treatment turns out to be effective in the prevention of clinical NFI, the implementation of treatment for patients with evident subclinical neuropathy in clinical practice will be complicated. The methods to detect subclinical neuropathy used in this study (TSA II and Neurocare 2000) will not be available in the field, since the devices are expensive and conditions under which the assessments have to take place, a steady environmental temperature of C, are difficult to realize in tropical climates. Therefore, it is important to search for a cheaper, portable method to detect subclinical neuropathy that can be easily used in field clinics. However, also without a test to determine subclinical neuropathy the results of this study can be useful to improve treatment guidelines. The current prediction rule allows to distinguish leprosy patients with a high risk for developing NFI [30]. However, at the moment, providing prophylactic prednisolone treatment to this group of patients is considered unethical, as a significant number of patients will take prednisolone unnecessarily. With the information of the TENLEP trials we hope the prediction rule can be refined so administering prophylactic prednisolone is acceptable in certain, well defined groups. In conclusion, the TENLEP study will add to the current understanding of neuropathy due to leprosy and will provide better insight into the effectiveness of prednisolone treatment in the prevention and recovery of nerve function loss in leprosy patients. If this study shows the effectiveness of prednisolone in the prevention and recovery of NFI it will improve treatment 43

44 options and contribute therefore to the prevention of permanent sensory and/or motor nerve function loss in people affected by leprosy and hence prevent disabilities and deformities which will improve the lives of many of these patients. 44

45 References 1. van Brakel WH, Nicholls PG, Wilder-Smith EP, Das L, Barkataki P, Lockwood DN: Early diagnosis of neuropathy in leprosy comparing diagnostic tests in a large prospective study (the INFIR cohort study). PLoS Negl Trop Dis 2008, 2(4):e World Health Organization: WHO expert committee on leprosy. Volume 874th edition. World Health Organization technical report series; Geneva, WHO 1998: van Veen NH, Nicholls PG, Smith WC, Richardus JH: Corticosteroids for treating nerve damage in leprosy. A Cochrane review. Lepr Rev 2008, 79(4): van Brakel WH: Peripheral neuropathy in leprosy and its consequences. Lepr Rev 2000, 71(Suppl):S146 S World Health Organization: Weekly epidemiological record. Volume 86th edition. Geneva, WHO, 2011: Nienhuis WA, van Brakel WH, Butlin CR, van der Werf TS: Measuring impairment caused by leprosy: inter-tester reliability of the WHO disability grading system. Lepr Rev 2004, 75(3): Broekhuis SM, Meima A, Koelewijn LF, Richardus JH, Benbow C, Saunderson PR: The hand-foot impairment score as a tool for evaluating prevention of disability activities in leprosy: An exploration in patients treated with corticosteroids. Lepr Rev 2000, 71(3): Khambati FA, Shetty VP, Ghate SD, Capadia GD, Pai VV, Ganapati R: The effect of corticosteroid usage on the bacterial killing, clearance and nerve damage in leprosy: A prospective cohort study: Part 1 - Study design and baseline findings of 400 untreated multibacillary patients. Lepr Rev 2008, 79(2): Smith WC, Nicholls PG, Das L, Barkataki P, Suneetha S, Suneetha L, Jadhav R, Sundar Rao PS, Wilder-Smith EP, Lockwood DN, et al: Predicting neuropathy and reactions in leprosy at diagnosis and before incident events-results from the INFIR cohort study. PLoS Negl Trop Dis 2009, 3(8):e Shetty VP, Khambati FA, Ghate SD, Capadia GD, Pai VV, Ganapati R: The effect of corticosteroids usage on bacterial killing, clearance and nerve damage in leprosy; part 3 - study of two comparable groups of 100 multibacillary (MB) patients each, treated with MDT 1 steroids vs MDT alone, assessed at 6 months post - release from 12 months MDT. Lepr Rev 2010, 81(1): Walker SL, Nicholls PG, Dhakal S, Hawksworth RA, Macdonald M, Mahat K, Ruchal S, Hamal S, Hagge DA, Neupane KD, et al: A phase two randomised controlled double blind trial of high dose intravenous Methylprednisolone and oral prednisolone versus intravenous normal saline and oral prednisolone in individuals with leprosy type 1 reactions and/or nerve function impairment. PLoS Negl Trop Dis 2011, 5(4): e

46 12. van Brakel WH, Anderson AM, Withington SG, Croft RP, Nicholls PG, Richardus JH, Smith WCS: The prognostic importance of detecting mild sensory impairment in leprosy: A randomized controlled trial (TRIPOD 2). Lepr Rev 2003, 74(4): Sundar Rao PSS, Sugamaran DST, Richard J, Smith WCS: Multi-centre, double blind, randomized trial of three steroid regimens in the treatment of type-1 reactions in leprosy. Lepr Rev 2006, 77(1): van Brakel WH, Nicholls PG, Lockwood DN, Rao PS, Smith WC: A scale to assess the severity of leprosy reactions. Lepr Rev 2007, 78(2): Screening Activity Limitation and Safety Awareness Users Manual V manual_v1.1pdf.pdf. 16. Participation Scale Users Manual v cale/participation_scale_users_manual_v._6.0.pdf. 17. Britton WJ, Lockwood DNJ: Leprosy. Lancet 2004, 363(9416): Conn HO, Poynard T: Corticosteroids and peptic ulcer: Meta-analysis of adverse events during steroid therapy. J Int Med 1994, 236(6): Da Silva JAP, Jacobs JWG, Kirwan JR, Boers M, Saag KG, Inês LBS, De Koning EJP, Buttgereit F, Cutolo M, Capell H, et al: Safety of low dose glucocorticoid treatment in rheumatoid arthritis: Published evidence and prospective trial data. Ann Rheum Dis 2006, 65(3): Richardus JH, Withington SG, Anderson AM, Croft RP, Nicholls PG, van Brakel WH, Smith WC: Adverse events of standardized regimens of corticosteroids for prophylaxis and treatment of nerve function impairment in leprosy: results from the TRIPOD trials. Lepr Rev 2003, 74(4): Ridley DS, Jopling WH: Classification of leprosy according to immunity. A five-group system. Int J Lepr Mycobact Dis 1966, 34(3): Bell-Krotoski J: Pocket filaments and specifications for the semmes-weinstein monofilaments. J Hand Ther 1990, 3: Brandsma W: Basic nerve function assessment in leprosy patients. Lepr Rev 1981, 52(2): Brandsma JW, van Brakel WH: Protocol for motor function assessment in leprosy and related research questions. Indian J Lepr 2001, 73(2): Walker SL, Nicholls PG, Butlin CR, Nery JAC, Roy HK, Rangel E, Sales AM, Lockwood DNJ: Development and validation of a severity scale for leprosy type 1 reactions. PLoS Negl Trop Dis 2008, 2(12): e

47 26. Ebenso J, Fuzikawa P, Melchior H, Wexler R, Piefer A, Min CS, Rajkumar P, Anderson A, Benbow C, Lehman L, Nicholls P, Saunderson P, Velema JP: The development of a short questionnaire for screening of activity limitation and safety awareness (SALSA) in clients affected by leprosy or diabetes. Disabil Rehabil 2007, 29(9): van Brakel WH, Anderson AM, Mutatkar RK, Bakirtzief Z, Nicholls PG, Raju MS, Das- Pattanayak RK: The participation scale: measuring a key concept in public health. Disabil Rehabil 2006, 28(4): Smith WC, Anderson AM, Withington SG, van Brakel WH, Croft RP, Nicholls PG, Richardus JH: Steroid prophylaxis for prevention of nerve function impairment in leprosy: randomised placebo controlled trial (TRIPOD 1). BMJ 2004, 328(7454): Capadia GD, Shetty VP, Khambati FA, Ghate SD: Effect of corticosteroid usage combined with multidrugca therapy on nerve damage assessed using nerve conduction studies: A prospective cohort study of 365 untreated multibacillary leprosy patients. J Clin Neurophysiol 2010, 27(1): Croft RP, Nicholls PG, Steyerberg EW, Richardus JH, Withington SG, Smith WC: A clinical prediction rule for nerve function impairment in leprosy patients-revisited after 5 years of follow-up. Lepr Rev 2003, 74(1):

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49 Chapter 3 Normal threshold values for a monofilament sensory test in sural and radial cutaneous nerves in Indian and Nepali volunteers Inge Wagenaar Wim Brandsma Erik Post Jan Hendrik Richardus Leprosy Review (2014) 85, 1 13

50 Abstract Introduction The monofilament test (MFT) is a reliable method to assess sensory nerve function in leprosy and other neuropathies. Assessment of the radial cutaneous and sural nerve in addition to usually tested nerves can help improve diagnosis and monitoring of nerve function impairment (NFI). To enable the detection of impairments in leprosy patients, it is essential to know the monofilament threshold in normal subjects of these two nerves. Methods The radial cutaneous, sural, ulnar, median and posterior tibial nerves of 245 volunteers were tested. All nerves were tested at three sites on both left and right body side. Normal monofilament thresholds were calculated per test-site and per nerve. Results We assessed 490 radial cutaneous and 482 sural nerves. The normal monofilament was 2-g (Filament Index Number (FIN) 4.31) for the radial cutaneous and 4-g (FIN 4.56) for the sural nerve, although heavy manual labourers demonstrated a threshold of 10-g (FIN 5.07) for the sural nerve. For median and ulnar nerves, the 200-mg (FIN 3.61) filament was confirmed as normal while the 4-g (FIN 4.56) filament was normal for the posterior tibial. Age and occupation have an effect on the mean touch sensitivity but do not affect the normal threshold for radial cutaneous and sural nerves. Conclusion The normal thresholds for the radial cutaneous and sural nerves are determined at the 2-g (FIN 4.31) and the 4-g (FIN 4.56) filament respectively. The addition of the radial cutaneous and sural nerve to sensory nerve assessment may improve the diagnosis of patients with impaired sensory nerve functions. 50

51 Introduction In leprosy care, accurate diagnosis and monitoring of nerve function impairment (NFI) are crucial. With early diagnosis and treatment of NFI, severe peripheral nerve impairment, often resulting in disabilities, can be prevented. The main tests used for diagnosis and monitoring of NFI in clinical practice are voluntary muscle tests (VMTs), ball-point tests (BPTs) and monofilament tests (MFTs) on, primarily, hands and feet. In leprosy affected nerves, sensory function loss often precedes motor function loss [1,2]. MFTs are able to detect and monitor sensory nerve function loss accurately, using variation in pressure. They offer more sensitive results than BPTs which generally provide only a yes/no outcome [3]. Monofilament testing is quick, sensitive and reliable and, therefore, a useful method to diagnose and monitor nerve impairment over time [4-6]. Most studies that assess NFI in leprosy patients examine the ulnar, median and posterior tibial nerves because these nerves are responsible for sensation on the hand palms and foot soles, comprising areas of the body most prone to ulceration and burns because of loss of protective sensation. In addition to these three nerves, assessment of the radial cutaneous (innervating a part of the dorsal side of the hand (Figure 1A) and the sural nerves (innervating the lateral side of the foot (Figure 1B) might also prove informative. Impairments of the sensory radial cutaneous and sural nerves are rarely tested in clinical and research settings because these impairments have little functional effect on patients. However, including assessment of the radial cutaneous and sural nerve can yield additional information to improve the diagnosis and monitoring of NFI. The inclusion of the dorsum of the hands and feet in sensory testing procedures has been recommended by Wexler et al. (2007) and Kuipers et al. (1994) because lesions and sensory impairment were located frequently on this part of the hands and feet [7,8]. Furthermore, the ILEP Nerve Function Impairment and Reaction (INFIR) study indicates that the sural and radial cutaneous nerve play an important role in the diagnosis of NFI. In that study, newly diagnosed patients without NFI were followed for two years, during which the sural became impaired in 37.2% of patients tested with MFTs (2), and the radial cutaneous was found to be affected as often as the median nerve (8.8%). Khambati et al. (2009) also reported sensory impairment in 28% and 23% of the sural and radial cutaneous nerves, respectively, in newly diagnosed multibacillary (MB) patients. However, these studies omitted to establish normal values for the MFT thresholds in relation to these two nerves. In patients who are at risk of NFI, diagnosis of impairment is necessary before reaction treatment can be started. However, impairment can only be determined when the monofilament threshold of normal subjects is known. For MFTs, the normal threshold represents the filament felt by a certain percentage (usually 95 or 97.5%) of normal subjects (not affected by the disease). Studies in different Asian countries determined that the 200-mg (FIN 3.61) filament represents the normal threshold for the palm of the hand (median and ulnar nerves) while the 2- g (FIN 4.31) filament was normal for the plantar side of the forefeet (posterior tibial nerve) [9-11]. These studies provide for the diagnosis of NFI in median, ulnar and posterior tibial nerves in Asian patients. However, before the radial cutaneous and sural nerves can be reliably used in 51

52 the diagnosis and follow-up of NFI, the normal values must be known. In this study, we establish normal threshold values for the radial cutaneous and sural nerves in Indian and Nepali volunteers and provide recommendations for clinical practice. Additionally, we assess the median, ulnar and posterior tibial nerves in order to compare our results with previous normative studies. Methods Subjects Subjects were recruited from four leprosy hospitals, two in Nepal and two in India. In each country, one urban and one rural hospital was selected. Visitors accompanying patients, patients from other departments (e.g. dermatology), and hospital staff were asked to participate. Subjects were excluded when they suffered from conditions with neurological implications, such as diabetes mellitus. Nearly all persons invited were willing to participate. A total of 245 Indian and Nepali volunteers, not known to have a history of leprosy, were recruited for MFT assessment. This study to establish normal values, was part of the Treatment of Early Neuropathy in Leprosy (TENLEP) trials, for which ethical approval was obtained from the Indian Council of Medical Research and the Nepal Health Research Council. All subjects were informed about the goal and procedures of the study and verbally agreed to participate. Data collection Age, occupation and dominant hand were recorded for all subjects. Prior to analysis, occupation was grouped into three categories: office work (e.g. students, business people and drivers) manual labour (e.g. cleaners, housewives, nurses and tailors) and heavy manual labour (e.g. farmers, labourers and porters). Three age groups were constructed before analyses: 15-30, and Some subjects were interviewed to determine their smoking behaviour (n=170); and habits of wearing footwear and types of footwear (closed shoes, sandals or a combination) (n=114), and whether and how often they sit cross-legged (n=116). Monofilaments The MFT is a touch sensibility test which indents the skin surface with a series of standard nylon filaments of same length but with varying degrees of thickness. A standard pocket set of six coloured Semmes-Weinstein monofilaments was used (Sorri-Bauru, São Paulo, Brazil), with bending forces ranging from 70-mg (green, FIN 2.81) to 300-g (pink, FIN 6.65) (Table 1) [12]. Test procedure According to standard protocol [13], filaments were applied at a straight angle to the skin and with sufficient force to make the filament bend slightly (C shape). The filaments were applied 52

53 slowly, the pressure maintained briefly and then removed slowly, taking approximately one second for each step. The subjects were asked to keep their eyes closed during this procedure and to indicate the place where they had felt the touch. Deviation of roughly two centimetres from the exact touch location was considered acceptable when there was adequate response time. The filaments were applied in sequential order from lightest (70-mg) to heaviest (300-g), applying the 70-mg (FIN 2.83) and 200-mg (FIN 3.61) filaments with a maximum of three times per site when the monofilament slipped or was not detected by the subject. The same applied for the 2-g (FIN 4.31) filament used on the feet. In order to keep the subjects focused, efforts were made to keep the test room silent and fans were switched off to minimize noise and the effect of the circulating air on the skin. A pre-test was carried out on all subjects before outcomes were recorded to make sure they understood the test. All data was collected by one trained assessor. Table 1- Characteristics of the standard pocket set of Semmes-Weinstein monofilaments Filament Filament index number Bending force (grams) Green Blue Purple Red Orange Pink Note: The filament index number is calculated from the bending force in milligram: FIN = log (force *10) Test sites In addition to the radial cutaneous and sural nerves, the ulnar, median and posterior tibial nerves were tested. For each nerve, three test-sites were assessed on both the left and right side of the body. For the ulnar, median and posterior tibial nerves, the same sites were examined as in the INFIR study [14]. However, we made one exception to the INFIR methodology by assessing the tip of the middle finger as the third point for the median rather than the index finger. The three test-sites for the radial cutaneous and sural nerves were selected as shown in Figure 1. The radial cutaneous nerve innervates a part of the dorsal surface of the hand: the proximal radial site of the index finger and thumb web space. This is shown in grey in Figure 1A [15]. The sural nerve innervates part of the plantar lateral surface of the foot and the lateral aspect of the ankle. The three test-sites can be found on the plantar lateral surface in one straight line about 1.5 cm above the sole of the foot (Figure 1B). 53

54 Figure 1 A- Dorsal innervation area of radial cutaneous nerve and the three test-sites. Figure 1 B- Innervation area of the sural nerve and the three test-sites Outcome For every test-site, the lightest filament detected was recorded, using a scoring method. The lightest filament (70-mg, FIN 2.83) was given a score of 0, and the score increased with one point for each thicker filament felt, with score 1 for 200-mg (FIN 3.61), 2 for 2-g (FIN 4.31), 3 for 4-g (FIN 4.56) and 4 for the heaviest, 10-g, filament (FIN 5.07). A patient is diagnosed with NFI when the overall score of a nerve from the total of the three test-sites is 3 or higher [14,16-18]. Weights and FINs are used to describe the filaments in this study because weights are well understood in the field and FINs are often used in scientific studies [10,19-22]. The normal threshold value for a test-site is defined as the monofilament that was detected by 95% of the volunteers, indicating that subjects only able to detect filaments heavier than the threshold are very likely to have NFI. Figure 2 shows an example in which 2-g, the first filament detected by at least 95% of the subjects, represents the normal threshold value. From the three individual test-sites, the overall normal value for the nerve is derived. The heaviest monofilament detected in any of the three sites determines the normal value for the entire nerve. 54

55 0.07 g 0.2 g 2 g 4 g 10 g 79%* 93% 98% 100% 100% * percentage of subjects detecting filament Normal threshold value is 2-g (FIN 4.31): the minimum of 95% is reached at this filament Figure 2- Explanation of the determination of our 95% normal threshold value (an example) Data analysis Normal monofilament thresholds were calculated per test-site and per nerve. A McNemar test for correlated proportions was carried out to test whether the percentage of subjects detecting the 200-mg and 2-g filament differed between the right and left hand and foot respectively. When no difference was found, the 95% normal thresholds were calculated for all nerves. Mann-Whitney tests were used to explore the association of sex, country of origin and dominant hand on mean FIN, calculated by averaging the three test-sites per nerve. Kruskal-Wallis tests were carried out to analyse the effect of age and occupational group on the mean FIN. Furthermore, the effect of footwear types and cross-legged sitting on the average filament detected by both nerves in the feet (sural and posterior tibial) was examined in a subgroup of volunteers using Kruskal-Wallis tests. Based on the assumption of independent data, all univariate analyses are performed on the nerves of the right body side only. A p-value of less than 5% was considered statistically significant. All tests were done using SPSS statistical package Results Radial cutaneous and sural nerves were assessed in 119 men and 126 women. Ulnar, median and posterior tibial nerves were tested in 198 of these persons. The mean age of the subjects was 35.5 years. Table 2 gives an overview of the characteristics of the 245 volunteers. The weight of the monofilament detected by left and right body side did not significantly differ for any test-site. As a result, normal threshold values were calculated using 490 hands and feet, independent of body side. Normal thresholds, based on the 95% cut-off, are shown in Table 3 for the entire study group and separately for the three age groups because age is known to have a significant effect on touch sensitivity [10,22,23]. For the radial cutaneous and sural nerves, the normal threshold values differed between the three test-sites. In consequence, the heaviest filament was used to determine the normal threshold for the entire nerve. 55

56 Table 2- Subjects characteristics of 245 volunteers % Sex (male) 48.6 Age (years) Country (n = 245) Smoking (n = 174) Occupation (n =229) Dominant Hand (n = 218) Shoe wearing (n = 114) Cross leg sitting (n = 116) India 38.8 Nepal 61.2 No 81.6 Yes 18.4 Office 41.9 Manual 41.5 Heavy 16.6 Right 95.4 Left 4.6 Sandals 64.0 Shoes 19.3 Both 16.7 No 28.4 Sometimes 28.4 Often 43.1 This resulted in a normal threshold of 2-g (FIN 4.31) for the radial cutaneous and 4-g (FIN 4.56) for the sural. For both ulnar and median nerves, the 200-mg (FIN 3.61) represents the normal threshold while for the posterior tibial it was the 4-g (FIN 4.56) filament. Univariate analyses identified age as the only variable that showed significant different mean FIN between the groups across all nerves. The higher the age group, the higher the mean filament weight detected by the subjects (Figure 3). However, comparison of the normal thresholds for the entire study group and the three age groups (Table 3) showed that thresholds are barely affected by age. The youngest group (15-30 years) shows lower thresholds for the radial, sural and posterior tibial; the oldest group (45-60 years) has a higher threshold for the median nerve. Occupation was significantly associated with the touch sensitivity of all nerves except the radial cutaneous nerve (Table 4). Volunteers undertaking heavy manual labour detected a higher mean size monofilament than those undertaking office work and manual labour, indicating a lower touch sensitivity. Moreover, the heavy manual labour group had a higher normal threshold than the entire study group for sural, ulnar, median and posterior tibial nerves. We also found that for the sural nerve, wearing shoes and never sitting cross-legged resulted in a lower normal threshold, hence a higher touch sensitivity. 56

57 Discussion This study showed that the normal threshold for monofilament testing for the radial cutaneous is the 2-g (FIN 4.31) filament. For the sural nerve, this threshold is the 4-g (FIN 4.56) filament. Furthermore, we established that the 200-mg (FIN 3.61) filament is the normal threshold for the ulnar and median nerve, while the 4-g (FIN 4.56) filament represents the normal threshold for the posterior tibial nerve. In this study, we confirmed the threshold for the palm of the hand as found in previous normative studies in Asians. The 200-mg (FIN 3.61) filament was identified as normal value for the median and ulnar nerve in two studies in Nepal, using a threshold cut-off of 95% (9) and 97% [11]. A study in Thailand showed that 97% of the normal subjects detected a 3.84 filament (this filament is not used in our study and is placed between 200-mg (FIN 3.61) and 2-g (FIN 4.31)) [10]. Furthermore, these three studies agreed the 2-g (FIN 4.31) filament was the normal threshold for the plantar side of the foot (posterior tibial nerve). This was not confirmed by our findings that the 4-g (FIN 4.56) filament represents the normal threshold for this nerve. It is possible that certain subgroups were overrepresented in previous studies, lowering the thresholds. For example, in the study by Kets et al. (1996), 64% of their subjects were under the age of 30. With this age distribution, a lower threshold is to be expected because we found that the youngest age group (15-29) has a lower threshold for the posterior tibial nerve than the entire group. This might also apply to groups which are left-handed, wearing closed shoes or sit cross-legged sometimes. The earlier studies, however, did not report on these parameters. The threshold we established for the radial cutaneous was consistent with our expectations, based on studies in Nepal, Thailand and India [19]. We assumed that the touch sensitivity of the dorsum of the hand would be similar to the palmar side of the hand and would lie between 200- mg (FIN 3.61) and 2-g (FIN 4.31), resulting in a threshold of the 2-g (FIN 4.31) filament. The Indian study, testing 57 normal subjects, assessed one test-site for the radial cutaneous nerve, corresponding with our second test-site in the radial cutaneous [19]. However, the cut-off used to determine the normal threshold is not clearly defined in the Indian study. We calculated from their data that the 200-mg (FIN 3.61) filament was detected on the right and left hand by 94.7% and 98.2% respectively. With 95% cut-off used in our study, the normal threshold would then correspond with the 2-g (FIN 4.31) and 200-mg (FIN 3.61) filament respectively. The difference in threshold between the dorsal and palmar side of the hand may be related to the number of cutaneous nerve endings. The density of tactile receptors can be mapped in detail by a 2-point discrimination test. From this test, it is known that the fingertips and the palm of the hands have a higher density of nerve endings than the dorsum of the hands [24]. 57

58 Table 3- Monofilament thresholds per test-site and per nerve for all subjects and per age group All subjects Nerves Radial cutaneous Sural Ulnar Median Posterior tibial Sites* R1 R2 R3 S1 S2 S3 U1 U2 U3 M1 M2 M3 P1 P2 P3 Result per test-site Percentage feeling MF threshold Result per nerve Age groups (n=168) Result per test-site Percentage feeling MF threshold Result per nerve (n=198) Result per test-site Percentage feeling MF threshold Result per nerve (n=124) Result per test-site Percentage feeling MF threshold Result per nerve *calculated over left and right body side combined R1= Radial Cutaneous test-site 1, R2= Radial Cutaneous test-site 2, R3= Radial Cutaneous test-site 3, S1= Sural test-site 1, S2= Sural test-site 2, S3= Sural test-site 3, U1= Ulnar test-site 1, U2= Ulnar test-site 2, U3= Ulnar test-site 3, M1= Median test-site 1, M2= Median test-site 2, M3= Median test-site 3, P1= Posterior tibial test-site 1, P2= Posterior tibial test-site 2, P3= Posterior tibial test-site 3

59 Radial cutaneous R1 R2 R FIN Age 5.00 Sural S1 S2 S FIN Age Figure 3- Trend line mean Filament Index Number (FIN) for three test-sites for a radial cutaneous and sural nerves for right body side 59

60 The sural threshold established (red, FIN 4.56) was higher than expected. Given that there is no literature on normal thresholds for the sural nerve, we assumed that the lateral border of the foot would have a similar threshold as the plantar side of the foot, determined in previous studies as 2-g (FIN 4.31). Although the threshold was not as expected, our assumption was correct because the threshold for the plantar and the lateral side of the foot were similar. To determine whether we should implement different thresholds for different groups, we performed univariate analyses and compared the thresholds per group (e.g. males vs. females et cetera). The analyses revealed that age and occupation were important variables associated with touch sensitivity. The decreased sensitivity with increasing age was also reported in other normative studies [10,11,22]. Mitchell and Mitchell (2000) discuss that neural degeneration, reducing the number of touch receptors and slowing down conduction, is the probable cause. In the heavy manual occupational group, normal thresholds were one level higher for the sural, ulnar, median and posterior tibial nerve. A normative study in Thailand found the same pattern with heavy manual labour being associated with higher sensory thresholds [10]. Heavy manual workers, like farmers and labourers, might have thicker callus on hands and feet, leading to higher monofilament thresholds. This would also explain why the radial cutaneous threshold does not differ between the labour groups because the dorsum of the hand is not involved in manual work and is therefore not likely to develop callus. The monofilament threshold for the radial cutaneous and sural nerve, as established in this study, can be used for all persons, with exception of the heavy manual labourer group for whom a higher threshold for the sural nerve applies. Given the multi-centre, multi-country study design, our findings are generalizable and especially useful for Asian countries where leprosy is often still prevalent and where neuropathies will become more widespread with the rising diabetes prevalence [25]. However, there are some factors that should be taken into account when evaluating our results. First, we mainly assessed close contacts of leprosy patients, such as family and staff of the hospitals. Our volunteers could have been unknowingly infected with leprosy because we did not test for leprosy. Hence, the potential presence of NFI could have resulted in higher thresholds. Second, we had to take into account the habit of this population to take family and friends into the test rooms. As a result, and despite our efforts, it was not always possible to test the volunteer in silence. This could have led to diminished focus and higher thresholds. However, the habit of bringing family will also occur in clinical practice so that our results will be still acceptable to determine the NFI status in leprosy patients. Both factors may have led to an overestimation of the thresholds but overestimation is better than underestimation that might lead to unnecessary treatment. In addition, we intended to correct the outcomes for multiple important variables in a multivariate analysis. However, no reliable tests were available since the data is nested, not independent, not normally distributed and categorical. Finally, even though monofilament testing is an optimal method in clinical practice to quantify NFI, most leprosy programs do not have access to the relatively expensive monofilaments. However, we hope that the rising prevalence of diabetes will stimulate the search for low-cost sources of monofilaments. If this is the case, leprosy clinics will be able to benefit as well. 60

61 Table 4- Mean filament index number (FIN) ± SD per nerve for different variable groups for right body side Radial cutaneous Sural Ulnar Median Posterior tibial Age (n) (84) 3.04 # ± # ± ± ± # ± (99) 3.19 ± ± ± ± ± (102) *3.28 ±0.31 *4.05 ±0.38 *3.02 ±0.30 * ±0.35 *4.06 ±0.45 Sex (n) Male (119) 3.19 ± ± ± ± ±0.44 Female (126) 3.13 ± ± ± ± ±0.43 Dominance (n) Right (208) 3.16 ± ± ± ± ±0.44 Left (10) 3.19 # ±0.22 * ± ± ±0.27 *3.72 # ±0.42 Country (n) India (95) 3.16 ± # ± ± ± ±0.49 Nepal (150) 3.16 ± ± ± ± ±0.40 Occupation (n) Office (96) 3.18 ± ± ± ± ±0.40 Manual (95) 3.15 ± ± ± ± ±0.42 Heavy manual (38) 3.15 ±0.29 * ±0.41 * ±0.32 * ±0.35 * ±0.51 Smoking (n) No (142) 3.11 # ± ± ± ± ±0.44 Yes (32) 3.17 ±0.33 *3.97 ± ± ±0.29 *4.04 ±0.46 Shoe Ŧ (n) Sandals (73) 3.90 ± ±0.40 Shoes (22) 3.76 # ± # ±0.33 Both (19) *4.09 ± ±0.39 Cross leg Ŧ (n) No (33) 3.88 # ± ±0.39 Sometimes (33) 3.74 ± # ±0.43 Often (50) *4.00 ± ±0.35 * a significant (p<0.05) difference between groups, Ŧ results shown only for nerves in foot, # Normal threshold is one FIN lower than the threshold for total group 1 Normal threshold is 2-g (FIN 4.31) where the total group threshold is 200-mg (FIN 3.61), 2 Normal threshold is 10-g (FIN 5.07) where the total group threshold is 4-g (FIN 4.56)

62 Similar to large studies on leprosy neuropathy (INFIR, TENLEP), three test-sites per nerve were assessed so that it was possible to use the scoring system described by Anderson [14,16,18]. Despite this, we chose to present our final results per nerve. The monofilament test is a quick and simple field method, and it is desirable to work with a minimal number of threshold values to guarantee its effectiveness. When the threshold differed between the three test-sites, we decided that the heaviest filament would determine the normal threshold. In those cases that the threshold differed between the test-sites, we could also have chosen alternative methods. For example by taking the filament detected in two out of three test-sites as the final threshold. However, in that case the thresholds would still be the same for the entire study group. When we would have taken the lightest filament of the three test-sites as threshold for the nerve, the threshold for the radial cutaneous and sural nerve would have been one filament lower (200-mg (FIN 3.61) and 2-g (FIN 4.31) respectively). With a lower threshold, NFI will be detected earlier but more false positives would occur. For this reason, we believe that it is better to use a threshold that is on the safe side for the radial cutaneous and the sural nerve, particularly as injuries due to loss of protective sensation are less common on the dorsum of hands and feet. Conclusions Taking into account all considerations, we recommend including the assessment of the radial cutaneous and sural nerves for monofilament testing in routine clinical practice. Testing the sensory function of the dorsum of the hand and lateral foot can give additional information on NFI, especially since these two nerves seem to be often impaired and are some of the earliest nerves impaired in leprosy [26]. Additional assessment of the radial cutaneous and sural nerves can improve diagnosis and monitoring of NFI, and may help prevent severe NFI and disabilities in patients with leprosy and neuropathies caused by other diseases (e.g. diabetes). In conclusion, the 2-g (FIN 4.31) filament should be taken as the normal threshold when testing the radial cutaneous nerve while the 4-g (FIN 4.56) filament is the normal threshold for the sural nerve. As an exception, heavy manual labourers have a higher threshold for the sural nerve, namely the 10-g (FIN 5.07) filament. 62

63 References 1. Khambati FA, Shetty VP, Ghate SD, Capadia GD. Sensitivity and specificity of nerve palpation, monofilament testing and voluntary muscle testing in detecting peripheral nerve abnormality, using nerve conduction studies as gold standard; a study in 357 patients. Lepr Rev Mar;80(1): van Brakel WH, Nicholls PG, Wilder-Smith EP, Das L, Barkataki P, Lockwood DN. Early diagnosis of neuropathy in leprosy--comparing diagnostic tests in a large prospective study (the INFIR cohort study). PLoS Negl Trop Dis. 2008;2(4):e Koelewijn LF, Meima A, Broekhuis SM, Richardus JH, Mitchell PD, Benbow C, et al. Sensory testing in leprosy: comparison of ballpoint pen and monofilaments. Lepr Rev Mar;74(1): Anderson AM, Croft RP. Reliability of Semmes Weinstein monofilament and ballpoint sensory testing, and voluntary muscle testing in Bangladesh. Leprosy Review. 1999;70(3): Naafs B, Dagne T. Sensory testing: a sensitive method in the follow-up of nerve involvement. Int J Lepr Other Mycobact Dis Oct-Dec;45(4): van Brakel WH, Khawas IB, Gurung KS, Kets CM, van Leerdam ME, Drever W. Intra- and inter-tester reliability of sensibility testing in leprosy. Int J Lepr Other Mycobact Dis Sep;64(3): Kuipers M, Schreuders T. The predictive value of sensation testing in the development of neuropathic ulceration on the hands of leprosy patients. Lepr Rev Sep;65(3): Wexler R, Melchior H. Dorsal sensory impairment in hands and feet of people affected by Hansen's disease in Israel. Lepr Rev Dec;78(4): Anderson AM, Van Brakel WH. Age specific normal thresholds for sensibility testing with monofilaments in a Nepali population. International Journal of Leprosy and Other Mycobacterial Diseases. 1998(66):69A. 10. Birke JA, Brandsma JW, Schreuders TAR, Piefer A. Sensory testing with monofilaments in Hansen's disease and normal control subjects. International Journal of Leprosy and Other Mycobacterial Diseases. 2000;68(3): Kets CM, Van Leerdam ME, van Brakel WH, Devillé W, Bertelsmann FW. Reference values for touch sensibility thresholds in healthy Nepalese volunteers. Leprosy Review. 1996;67(1): Bell-Krotoski J. "Pocket" filaments and specifications for the Semmes-Weinstein monofilaments. J Hand Ther. 1990;3:

64 13. Bell-Krotoski J, Weinstein S, Weinstein C. Testing sensibility, including touch-pressure, two-point discrimination, point localization, and vibration. J Hand Ther Apr- Jun;6(2): van Brakel WH, Nicholls PG, Das L, Barkataki P, Suneetha SK, Jadhav RS, et al. The INFIR Cohort Study: investigating prediction, detection and pathogenesis of neuropathy and reactions in leprosy. Methods and baseline results of a cohort of multibacillary leprosy patients in north India. Lepr Rev Mar;76(1): Campero M, Serra J, Ochoa JL. Peripheral projections of sensory fascicles in the human superficial radial nerve. Brain. 2005;128(4): Anderson A, Reed N, van Brakel W, Croft RP. Optimisation of a monofilament test for sensibility for use in a field programme of prevention of impairment. International Journal of Leprosy and Other Mycobacterial Diseases. 1998(66):69A. 17. van Brakel WH, Shute J, Dixon JA, Arzet H. Evaluation of sensibility in leprosy-- comparison of various clinical methods. Lepr Rev Jun;65(2): Wagenaar I, Brandsma W, Post E, van Brakel W, Lockwood D, Nicholls P, et al. Two randomized controlled clinical trials to study the effectiveness of prednisolone treatment in preventing and restoring clinical nerve function loss in leprosy: the TENLEP study protocols. BMC Neurology DCOM ( (Electronic)). 19. Malaviya GN, Husain S, Girdhar A, Girdhar BK. Sensory functions in limbs of normal persons and leprosy patients with peripheral trunk damage. Indian Journal of Leprosy. 1994;66(2): Jeng C, Michelson J, Mizel M. Sensory thresholds of normal human feet. Foot Ankle Int Jun;21(6): McPoil TG, Cornwall MW. Plantar tactile sensory thresholds in healthy men and women. Foot. 2006;16(4): Mitchell PD, Mitchell TN. The age-dependent deterioration in light touch sensation on the plantar aspect of the foot in a rural community in India: Implications when screening for sensory impairment. Leprosy Review. 2000;71(2): van Brakel W. Peripheral Nerve Function Assessment. Surgical Reconstruction & Rehabilitation in Leprosy and other Neuropathies. Nepal: Ekta Books; p Nolan MF. Two-point discrimination assessment in the upper limbs in young adult men and women. Phys Ther. 1982;62(7): Wild S, Roglic G, Green A, Sicree R, King H. Global Prevalence of Diabetes: Estimates for the year 2000 and projections for Diabetes Care. 2004;27(5): van Brakel WH, Nicholls PG, Das L, Barkataki P, Maddali P, Lockwood DN, et al. The INFIR Cohort Study: assessment of sensory and motor neuropathy in leprosy at baseline. Lepr Rev Dec;76(4):

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67 Chapter 4 Reliability of clinical nerve function assessment in peripheral neuropathies Wim Brandsma Inge Wagenaar Erik Post Peter Nicholls Jan Hendrik Richardus Leprosy Review (2014) 85, 29 35

68 Abstract Introduction Sensory and/or motor nerve function impairment as a consequence of neuropathy is often assessed using electro-neurophysiological tests. However, in low resource countries where the required equipment is rarely available, manual muscle strength testing (MMST) and monofilament testing (MFT) offer very reliable alternatives. In six leprosy programmes in four Asian countries, a multi-centre randomised clinical trial (RCT) was carried out to assess the effect of corticosteroids on neuropathy in leprosy-affected people. The sensory and motor nerve function was tested using MMST and MFT, including new test sites for the sural and radial cutaneous nerves (MFT) and the posterior tibial and common peroneal nerves (MMST). The reliability studies of the MMST and MFT tests of the TENLEP (Treatment of Early Neuropathy in LEProsy) trials are presented here. Methods Two assessors in each centre independently used the MFT and MMST in 30 leprosy-affected people. Results Reliability is good to very good for MFT in nearly all nerves. MMST also shows good to very good agreement, with a few exceptions. Conclusion Our study confirms that MMST and MFT can be performed reliably, and that the new tests also have acceptable reliability. 68

69 Introduction There are many neuropathies that may result in sensory and/or motor function impairment that can be assessed and evaluated with practical clinical techniques such as manual muscle strength testing (MMST) a and monofilament testing (MFT) [1]. Electro-neurophysiological assessments have an important place in the differential diagnosis and follow up of suspected neurological diseases but are very rarely available in low-resource countries. When clinicians are trained, MMST and MFT could still be a useful clinical adjunct to the electro-neurophysiological assessments. Various studies have reported good reliability of both MMST and MFT [2-8]. Many of these studies have been conducted in leprosy-endemic countries because of the availability of large numbers of subjects which makes it easier to conduct such studies in a relatively short time. MMST and MFT, however, could also be very useful in assessing neuropathies due to other common diseases such as diabetes, or neuromuscular diseases such as hereditary motor and/or sensory neuropathies. This study is in part a replicate study of reliability testing. The difference, however, is that this is a multi-centre reliability study that includes sensory and motor tests at sites for which reliability had not yet been determined. In a randomised clinical trial (RCT), studying the efficacy of corticosteroids in the prevention and treatment of leprosy neuropathy, MMST and MFT were used to screen patients for intake, and for the follow-up of patients for the duration of the trials [9]. TENLEP (Treatment of Early Neuropathy in LEProsy) consists of two related multicentre, multicountry trials. In the Sub-clinical trial, patients with normal monofilament and muscle strength values, but with impaired electrodiagnostic or thermodiagnostic parameters, are enrolled. The outcomes for a group that will receive corticosteroids for 20 weeks will be compared with the outcome for a placebo group. The aim is to assess whether corticosteroids may prevent the onset of clinically detectable nerve function loss. Patients with impaired nerve function of less than 6 months duration, as confirmed by MMST and MFT, are enrolled in the other arm of the study: the Clinical trial. In this trial, the currently recommended corticosteroid treatment for reaction is compared with an alternative regimen that is 3-4 months longer. The protocols for both trials have been published [9]. As decisions for enrolment and follow-up in both trials are based on the results of MMST and MFT, we felt that these assessments needed to show good reliability in and between the participating study centres. Hence, for both studies, we determined the reliability of MMST and MFT. a VMT (Voluntary Muscle Testing) is the more commonly accepted abbreviation for muscle testing. However, voluntary muscles can also be assessed with dynamometers. To distinguish between the two, the first author prefers to add Manual. 69

70 Methods Following approval by local and national ethical committees, reliability was assessed in the six study centres where TENLEP is implemented: two each in India and Nepal and one centre each in Indonesia and Bangladesh. The level of experience of selected staff varied between and within centres; therefore all participating staff were trained to perform testing in a standard manner. For this purpose, a Standard Operational Procedure (SOP) manual was developed. Test-sites and procedures Table 1 shows nerves in which motor and sensory function are commonly affected and assessed in the diagnosis and follow up of leprosy neuropathy. Table 1- Overview of nerves and their test-sites for MMST and MFT Nerve Muscle strength testing Sensory testing with monofilaments Facial Eye closure - Ulnar Little finger abduction Little finger/ hypothenar Median Thumb abduction Thumb/ middle finger Radial (cutaneous) Wrist extension Radial site index/ thumb web Common Peroneal (CP) Foot dorsiflexion - Deep branch CP Great toe extension - Posterior tibial Great toe plantar flexion Great toe plantar surface/ forefoot Sural - Lateral border foot Motor For each nerve with motor function, tests on one muscle group stimulated by that nerve were performed, using the Medical Research Council (MRC) grades [1]. Motor function tests for facial, ulnar, median, radial cutaneous and common peroneal were carried out as described elsewhere [1]. As a relatively new test we introduced the great toe up and down test to assess the motor function of the terminal branch of the common peroneal nerve, and the posterior tibial nerve respectively. The great toe up test is performed when the patient is sitting with his/her feet flat on the ground. The patient is asked to lift the great toe only and the assessor determines muscle strength, applying pressure on the proximal phalanx. The great toe down test is also called the paper grip test [10]. For this, an MRC score of 5 (strong), 4 (weak) or 0 (paralysed) could be given. 70

71 Sensory For each nerve with a sensory component, three test-sites were used within the innervation area of that nerve. Monofilament testing was performed using five monofilaments: 200 mg, 2 g, 4 g, 10 g and 300 g. For the foot, because of known higher thresholds, the lightest, 200 mg filament, was omitted. Each of three sites was then given a score based on the filament felt, the heaviest monofilament getting the highest score, allowing a maximum total score of 15 for the ulnar, median and radial nerves (4 filaments) and 12 for the foot (3 filaments). For the ulnar, median and posterior tibial nerves, the same sites were examined as in the INFIR study [11], except for the median nerve where we used the tip of the middle finger instead of the index finger. For the first time, the radial cutaneous and sural nerves were also assessed using three test-sites, which are shown in Figure 1. Figure 1- Test-sites for radial cutaneous (hand) and sural nerve (foot) monofilament testing Reliability testing To develop expertise, a large number of patients were assessed under supervision of experienced assessors before the reliability study was initiated. Following that period of skill development, both therapists were asked to randomly and independently assess a minimum of 30 subjects, all of whom were leprosy-affected people. The testers were blind to each other s results and the order of subjects was changed randomly between testers. Motor and sensory nerves were assessed bilaterally, effectively doubling the number of tests. Test results of both assessors were sent to, and analysed by the same statistician (PN). 71